sensors Article
Theoretical and Experimental Comparison of Different Formats of Immunochromatographic Serodiagnostics Dmitriy V. Sotnikov, Anatoly V. Zherdev
ID
and Boris B. Dzantiev *
A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia;
[email protected] (D.V.S.);
[email protected] (A.V.Z.) * Correspondence:
[email protected] or
[email protected]; Tel.: +7-495-9543142 Received: 10 November 2017; Accepted: 22 December 2017; Published: 25 December 2017
Abstract: In this study, a comparative theoretical and experimental analysis of two immunochromatographic serodiagnostics schemes, which differ in the immobilization of immunoreagents and the order of the formation of immune complexes, is performed. Based on the theoretical models, the assays are characterized to determine which scheme has a higher quantity of the detected complex and thus ensures the sensitivity of the analysis. The results show that for the effective detection of low-affinity antibodies, the scheme involving the immobilization of the antigen on gold nanoparticles and the antibody-binding protein on the test strip was more sensitive than the predominantly used scheme, which inverts the immunoreagents’ locations. The theoretical predictions were confirmed by the experimental testing of sera collected from tuberculosis patients. Keywords: lateral flow immunoassay; detection of antibodies; mathematic simulation; tuberculosis
1. Introduction Immunochemical test systems are widely used in medical and veterinary diagnostics [1]. Serodiagnostics is a form of immunoassay used to detect antibodies specific to the infectious agent in the blood. These antibodies are generated in response to the presence of an infectious agent in the body, and detecting them is often more informative than detecting the pathogen [2,3]. Immunochromatographic serodiagnostics are conducted using lateral flow test strips, which are also especially promising for mass use. Testing can be conducted outside the laboratory because it does not require equipment or highly qualified personnel. Importantly, the results can be obtained in 10–15 min with this method [4–7]. Because of their rapidity, methodological simplicity and low cost, immunochromatographic assays (ICA) are widely used in medical and veterinary diagnostics. In addition, the above advantages make serodiagnostic ICA an effective tool for evaluating the results of immunization of humans and experimental animals. The traditional scheme for immunochromatographic serodiagnosis (i.e., scheme A) is shown in Figure 1A. In this scheme, a marker (usually gold nanoparticles) is conjugated with an immunoglobulin-binding protein (e.g., anti-species antibodies, staphylococcal protein A, or streptococcal protein G). A sample of blood or serum then flows along the membranes of the test strip. In the first stage, all immunoglobulins in the sample interact with the conjugate. Next, the liquid reaches the zone of the strip with the immobilized antigen (i.e., the analytic zone). The conjugate interacts with the antigen molecules, resulting in the formation of a colored complex containing gold nanoparticles, immunoglobulin-binding protein molecules, immunoglobulins from the sample, and immobilized antigen. The given scheme is used in the most of lateral flow tests described in the literature [8–12], as well as in the most of the manufactured serodiagnosis tests such as the products of the companies AmeriTek (Everett, WA, USA; www.ameritek.org), Dr. Fooke Laboratories (Neuss, Sensors 2018, 18, 36; doi:10.3390/s18010036
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Serodiagnostic ICA may be realized in other formats that differ in the order of detectable complex formation and, accordingly, in the composition of the colored complex in the analytic zone [13–16]. The most popular alternate format of serodiagnostic ICA is a scheme (scheme B) in which the antigen molecules are conjugated to gold nanoparticles, and the immunoglobulin-binding protein is 18, immobilized on the test strip (Figure 1B). In this ICA, specific immunoglobulins2 first Sensors 2018, 36 of 15 interact with the antigen. The gold nanoparticles conjugate, form complexes, then migrate to the analytic zone and bind there. Both kinds of test strips also contain a second control zone with Germany; www.fooke-labs.de), Human (Wiesbaden, Germany; www.human.de), Standard immobilized antibodies (anti-species ones in scheme A and antibodies against the antigen forDiagnostics scheme B) (Yongin-si, Korea; www.standardia.com), Vedalab (Alençon, France; www.vedalab.com), etc. to control the strips’ functionality.
Traditional scheme (A)alternate and alternate B (B) of immunochromatographic Figure 1.1.Traditional scheme A (A)Aand scheme Bscheme (B) of immunochromatographic serodiagnosis. serodiagnosis.
Serodiagnostic ICA may be realized in other formats that differ in the order of detectable complex Sotnikov al. [16] showed thatcomposition in some cases, B allowed increased diagnostic sensitivity formation and,etaccordingly, in the of scheme the colored complex in the analytic zone [13–16]. of the analysis. However, it is not known whether scheme B is always characterized by a higher The most popular alternate format of serodiagnostic ICA is a scheme (scheme B) in which the sensitivity than scheme AA, or if this is true only under certain Moreover, the causes of antigen molecules are conjugated to gold nanoparticles, and theconditions. immunoglobulin-binding protein theimmobilized change in sensitivity when an (Figure alternate1B). scheme is used is on the test strip In this ICA,remain specificunexplained. immunoglobulins first interact To address these issues, the present study simulates the processes ofthen immune interactions in two with the antigen. The gold nanoparticles conjugate, form complexes, migrate to the analytic ICA schemes and compares their characteristics. In a recent study [17], a mathematical model of a zone and bind there. Both kinds of test strips also contain a second control zone with immobilized serodiagnostic ICA with theintraditional (i.e.,antibodies A) scheme was proposed. B has notcontrol been antibodies (anti-species ones scheme A and against the antigenScheme for scheme B) to previously described using mathematical tools. The development and analysis of this model are the strips’ functionality. provided for the first time in thisthat article. In addition, the obtained model is compared with sensitivity the model Sotnikov et al. [16] showed in some cases, scheme B allowed increased diagnostic of the traditional scheme A, and the advantages of each analytical scheme are then justified. The of the analysis. However, it is not known whether scheme B is always characterized by a higher potential of scheme B to increase sensitivity is confirmed experimentally using the serodiagnosis of sensitivity than scheme AA, or if this is true only under certain conditions. Moreover, the causes of the one of the most significant diseases: tuberculosis. change in sensitivity when infectious an alternate schemehuman is usedpulmonary remain unexplained. To address these issues, the present study simulates the processes of immune interactions in two ICA schemes and compares their characteristics. In a recent study [17], a mathematical model of a serodiagnostic ICA with the traditional (i.e., A) scheme was proposed. Scheme B has not been previously described using mathematical tools. The development and analysis of this model are provided for the first time in this article. In addition, the obtained model is compared with the model of the traditional scheme A, and the advantages of each analytical scheme are then justified.
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The potential of scheme B to increase sensitivity is confirmed experimentally using the serodiagnosis of one of the most significant infectious diseases: human pulmonary tuberculosis. 2. Materials and Methods 2.1. Reagents 3,30 ,5,50 -Tetramethylbenzidine (TMB), chloroauric acid, bovine serum albumin (BSA), and sodium azide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant 38-kDa antigen (Rv0934) of Mycobacterium tuberculosis was obtained from Arista Biologicals (product code AGMTB-0220, Allentown, PA, USA), and recombinant staphylococcal protein A was obtained from Imtek (Moscow, Russia). All salts and other additional reactants were of analytical or reagent grade. The Simplicity purification system (Millipore, Bedford, MA, USA) was used for water deionization. Serum samples from healthy donors were supplied by the State Scientific Center, Institute of Immunology (Moscow, Russia). Serum samples from persons with a confirmed case of pulmonary tuberculosis were supplied by Dr. V. G. Avdienko, Central Tuberculosis Research Institute (Moscow, Russia). In all the serum samples, the presence of anti-mycobacterial antibodies was confirmed by quantitative immunoenzyme assay (see below). 2.2. Immunoenzyme Assay of Specific Antibodies in Serum Samples Antigen Rv0934 was adsorbed in a 96-well Greiner microplate (100 µL per well) for 16 h at 4 ◦ C from a 1 µg/mL solution in 50 mM Na-carbonate buffer (50 mM, pH 9.6). The wells were washed with a K-phosphate buffer (50 mM, pH 7.4), with 0.1 M NaCl and 0.05% detergent Triton X-100 (PBST). Then, of sera 100-fold diluted with PBST was added (100 µL per well) and incubated for 1 h at 37 ◦ C. The microplate was washed, and monoclonal antibodies against human IgG labeled with horseradish peroxidase were added (160 ng/mL, 100 µL in PBST) for a 1-h incubation at 37 ◦ C. After washing, the activity of the bound peroxidase was tested by the addition of 0.4 mM TMB in an Na-citrate buffer (100 mM, pH 4.0), with 0.006% H2 O2 (100 µL per well), incubated for 15 min, and measured for its OD450 values (optical density at 450 nm). 2.3. Synthesis of Gold Nanoparticles The Frens method [18] was used for the synthesis: 0.2 mL of 5% chloroauric acid was added to 97.5 mL of water and heated to boiling. After this, 1.5 mL of 1% sodium citrate was added. The reactants were boiled for 25 min and then cooled. Size of the obtained gold nanoparticles was characterized using CX-100 transmission electron microscope (Jeol, Akishima-shi, Japan) and Image Tool software (University of Texas Health Science Center, San Antonio, TX, USA) as described in [16]. An average diameter of the particles was 28 ± 3 nm. 2.4. Conjugation of Protein A and Antigen Rv0934 with Gold Nanoparticles Firstly, the flocculation curves were obtained for mixtures of the mycobacterial antigen Rv0934 and the staphylococcal protein A with gold nanoparticles as described in [19]. The protein solutions of different concentrations were added to gold nanoparticles preparation, 10% NaCl was then added and OD590 of the mixture was measured after 10-min incubation at room temperature. The plateaus of the obtained concentration dependences are considered as zones of the saturation of gold nanoparticles surface by immobilized antibodies. The given saturations were reached for the concentrations equal to 8 µg/mL for the antigen Rv0934 and 20 µg/mL for the protein A, and the given concentrations were chosen for the conjugation. The antigen Rv0934 and the protein A were dialyzed against an Na-carbonate buffer (10 mM, pH 9.0) and diluted to the concentrations indicated above. The pH value of the gold nanoparticle preparation with OD520 equal to 1.0 was shifted to 9.0 using a 0.1 M K2 CO3 and were then added
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to the antigen Rv0934 and the protein A solutions. The mixture was then stirred for 10 min, and a BSA was added to the final concentration of 0.25%. Finally, the gold nanoparticles with the bound proteins were separated by centrifugation (8000 g, 20 min) and redissolved in PBS with 0.25% BSA. The conjugates were stored at 4 ◦ C with the addition of sodium azide (0.05%) for long-term storage. 2.5. Application of Reagents onto Immunochromatographic Membranes All membranes and pads were obtained from Advanced Microdevices, Ambala Cantt, India. An IsoFlow dispenser (Imagene Technology, Hanover, NH, USA) was used for the reagents application. In the case of scheme A, the analytic zone was formed by applying the antigen Rv0934 of M. tuberculosis water solution (1.0 mg/mL) onto the nitrocellulose membrane CNPH90, 2 µL of the antigen solution per 1 cm. The conjugate of protein A and gold nanoparticles was applied to the conjugate release pad PT-R5 at a dilution corresponding to OD520 = 10.0 in a volume of 11 µL per 1 cm. In the case of scheme B, the analytic zone was formed by applying the protein A onto the nitrocellulose membrane CNPH90, 2 µL of its solution (10 mg/mL in K-phosphate buffer (50 mM, pH 7.4)) was applied per 1 cm. The conjugate of antigen Rv0934 and gold nanoparticles was applied to the conjugate release pad PT-R5 at a dilution corresponding to OD520 = 2.0 in a volume of 8 µL per 1 cm. 2.6. Preparation of Immunochromatographic Test Strips The membranes with the applied reagents were air dried for one day. The test strips were prepared using the nitrocellulose membrane, conjugate release pad, a sample pad FR1(0.6), and a final adsorbent pad, AP045 as proposed in [17]. The multimembrane composite was assembled and then cut using an Index Cutter-1 automated guillotine cutter (A-Point Technologies, Gibbstown, NJ, USA) into strips 3.5 mm wide. The strips were packed in laminated aluminum foil bags using a FR-900 conveyor (Dingli Packing Machinery, Wenzhou, China) with added silica gel as the desiccant. The preparation of the test strips was carried out in a room with relative humidity
